Directed protein evolution is a powerful technology for protein engineering. It generally involves generating a library of protein variants and identifying those with desired properties by selection or screening. In terms of library complexity, in vitro selection methods are superior to cell-based selection methods by several orders of magnitude, allowing a much larger sequence space in proteins to be sampled. There is currently no effective in vitro method to select proteins with desired mutations that enhance their thermostability. Thermostable proteins can function at high temperatures, are generally robust and resistant to degradation under a variety of conditions, and therefore are valuable for a wide range of industrial and medical applications. This project intends to develop a technology for in vitro selection of protein thermostability. We plan to achieve this goal by (1) constructing a reconstituted in vitro protein synthesis system (thermo PURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 720C, and (2) applying such system for directed evolution of proteins with enhanced thermostability. We have successfully completed the first phase of the project (Phase I) and established an initial thermo PURE system that allowed in vitro synthesis of active full- length proteins at temperatures up to 600C. For the Phase II, we propose to commercialize the thermo PURE system by optimizing the system and testing more target proteins. We also propose to use the thermo PURE system in conjunction with in vitro selection technologies, such as in vitro compartmentalization and ribosome display, for directed evolution of thermostable proteins from their mesophilic origins. We plan to test several selection schemes of directed evolution for a variety of enzymes and proteins. If successful, this project would lead to the following unique and valuable commercial products: (1) a protein thermo synthesis kit; (2) a service for engineering proteins with enhanced thermostability; (3) thermostable nucleic acid enzymes as new reagents for research communities and thermostable single-chain antibodies for therapeutic applications.